Screening of a novel hepatic syndrome and its uses

ABSTRACT

The invention concerns methods of screening for a hepatic syndrome occurring in the young adult and associating cholesterol biliary microlithiasis, intrahepatic cholestasis and several mutations of the MDR3 gene. The invention is also directed to methods for the treatment of said syndrome. The hepatic syndrome screening methods comprise detecting, from a nucleic acid sample extracted from peripheral blood mononucleate cells, heterozygous mutations of the MDR3 gene and/or homozygous mutations of the MDR3 gene that do not eliminate the expression of the protein expressed by the MDR3 gene, which has phosphatidylcholine carrier activity, in adult subjects associating cholesterol biliary microlithiasis and intrahepatic cholestasis.

RELATED APPLICATIONS

This is a divisional of co-pending application Ser. No. 10/360,705,filed Feb. 10, 2003, which is a continuation-in-part of internationalapplication PCT/FR01/02553, filed Aug. 6, 2001 which claims priority toFrench application 00/10428, filed Aug. 8, 2000 All of the foregoingdocuments are hereby expressly incorporated by reference in theirentireties into the present application.

The present invention relates to the screening of a hepatic syndromeoccurring in young adults and associating (i) intrahepatic hyperechoicfoci with or without intrahepatic sludge or microlithiasis and furtheron a cholesterol microcholelithiasis, intrahepatic cholestasis and (ii)one or more mutations of the MDR3 gene (point mutations and/or SNPs).

The present invention also relates to the treatment of said syndrome.

Cholesterol cholelithiasis is characterized by the presence of calculiof cholesterol or of calcium bilirubinate. Cholesterol lithiasis is themost common form of cholelithiasis (80%). It is characterized by theformation of calculi of cholesterol in the gall bladder, due to anexcess of bile cholesterol compared to solubilizing molecules, namelybile acids and phospholipids. Its prevalence is estimated, taking allages into account, to be 10 to 20% of the population in industrializedcountries. It is more prevalent in women and increases considerably withage (40% in women over the age of 60). Gall bladder lithiasis can be asource of complications: cholecystitis, cancer of the gall bladder,migration of calculi in the common bile duct, a cause of angiocholitisand of pancreatitis. Treatment for this diseases is based mainly onsurgery: cholecystectomy by laparotomy or celioscopy, biliary drainagewith peroperative or perendoscopic extraction of the common bile ductcalculi. Medical treatment, which consists of administeringchenodeoxycholic acid and/or ursodeoxycholic acid, is reserved for formsof non-complicated cholesterol cholelithiasis of the gall bladder.

Chronic biliary cholestasis is characterized by a deficiency in passageof the bile from the liver to the extrahepatic bile ducts and theintestine. The causes and physiopathology are varied (Poupon et al.,2000, J. Hepatol., 32, 129-140). Most chronic biliary cholestases canprogress to biliary cirrhosis and hepatocellular insufficiency requiringa liver transplant.

In children for example, cholestasis is observed in biliary atresia, inAlagille syndrome or in progressive familial intrahepatic cholestasis(PFIC). In general, genetic abnormalities are observed in these variousdiseases.

Familial intrahepatic cholestasis (PFIC) are infantile recessivediseases which are characterized by intermittent jaundice, severecholestatis and cirrhosis, with a fatal outcome during the first tenyears of life.

These diseases manifest themselves in several clinical forms, two ofwhich are determined by mutations present on genes encoding ABCtransporters. Byler's disease, or progressive familial intrahepaticcholestasis type 1, which is characterized by normal cholesteremia andnormal serum γ-glutamyl transferase activity, was the first described.It is caused by mutation of the FIC1 gene, a type V ATPase involved inphospholipid transport from the outer wall to the inner wall of variouscell membranes. More specifically, PFIC type 1 is characterized byrecurrent episodes of jaundice, severe pruritis, normal γ-GT activityand normal cholesterol levels, high concentrations of bile acids in theserum and low levels of bile acids in the bile. The PFIC1 locus wasfound in chromosome 18q21-q22; five mutations were identified: adeletion, a deleted exon and three missense mutations; these missensemutations concern domains which are highly conserved in type V ATPases(E. Jacquemin et al., J. Hepatol., 1999, 31, 377-381).

Familial cholestasis type 2 exhibits the same clinical signs of Byler'sdisease, but it is caused by mutations in the FIC2 gene, which encodesthe BSEP (Bile Salt Export Pump) transporter. This gene is the homologof the murine SPGP gene, the product of which, expressed exclusively inhepatocytes, is involved in bile salt transport. PFIC type 3 ischaracterized by a severe pruritis, normal γ-GT activity and a normalcholesterol level in the serum, high concentrations of bile acids in theserum and very low levels of bile acids in the bile. The PIFC2 locus wasfound in chromosome 2q24: 10 mutations were identified, includingdeletions and missense mutations involving important domains of the BSEPtransporter (E. Jacquemin et al., J. Hepatol., 1999, 31, 377-381).

Individuals suffering from progressive familial intrahepatic cholestasistype 3 (PFIC3) have mutations in the MDR3 gene. PFIC type 3 differs fromthe other PFICs in particular by the fact that it has a high serumγ-glutamyl transferase activity. The MDR (Multi Drug Resistance) familyof ABC transporters is a multigene family of homologous proteins, someof which (MDR3) are not involved in drug resistance, and arephosphatidylcholine transporters. The MDR3 gene, revealed in 1993, is aphospholipid translocator. It is expressed in hepatocytes and providesphosphatidylcholine translocation, thus leading to secretion of thisphospholipid into the bile.

Mice in which the gene equivalent to MDR3 has been knocked out, mdr 2(−/−), exhibit a deficiency secretion of phospholipids into the bile,which causes physiological modifications close to those patientssuffering from progressive familial intrahepatic cholestasis type 3. Twopatients suffering from this disease and carrying mutations on both MDR3alleles have recently been described; the histopathological profile ofthe two patients is similar to that observed in the mdr 2 (−/−) mice (J.Marleen L. de Vree et al., PNAS, 1998, 95, 1, 282-287). Morespecifically, at the histological level, portal fibrosis, proliferationin the bile ducts and the presence of an inflammatory infiltrate areobserved. However, no cholesterol microcholelithiasis is observed inthese patients suffering from PFIC3 or in the mdr 2 (−/−) mice. At thegene level, in the first patient, a homozygous 7 bp deletion wasobserved, beginning at amino acid 132, leading to a reading frame shiftand introducing a stop codon, 29 codons downstream; the second patientis homozygous for a nonsense mutation in codon 957 (C/T) whichintroduces a stop codon (TGA): this mutation deletes the TaqIrestriction site. In such a pathological condition, truncated, nonactiveproteins, missing at least one ABC motif, are expressed. Furthermore,additional nonsense mutations and missense mutations, associated withvery low levels of bile phospholipids, have also been observed.

Cholestasis appearing only during pregnancy have also been observed inpatients exhibiting a heterozygous mutation of the MDR3 gene; in suchcases, no microlithiasis has been observed (E. Jacquemin et al., TheLancet, 1999, 353, 210-211; Dixon et al., Human Molecular Genetics,2000, 9, 1209-1217).

More specifically, in these patients, the following are observed:

-   -   either a heterozygous deletion of a nucleotide (1712delT)        beginning at amino acid 571, leading to a reading frame shift        introducing a stop codon, 15 codons downstream: this mutation,        which leads to the expression of a truncated, nonactive protein,        was detected in pregnant women from a family suffering from        PFIC3 (Jacquemin et al., The Lancet, 1999, mentioned above);    -   or a missense mutation in exon 14 (A546D): this mutation was        detected in pregnant women with no family history of PFIC (Dixon        et al., mentioned above).

Surprisingly, the inventors have found that, in young adults (20-40years old), pathological conditions exist which differ from PFIC type 3,which are triggered under situations such as pregnancy, the taking ofsex hormones, sustained fasting, sustained parenteral nutrition, andsurgical interventions, or by hepatotoxic cofactors (obesity, diabetes,the taking of medicines liable to modify the composition of bile:fibrates or diuretics, in particular), and in which the following areobserved in combination:

-   -   cholesterol microcholelithiasis    -   intrahepatic cholestasis and    -   at least one mutation of the MDR3 gene (point mutation and/or        SNPs).        The onset of this syndrome is related to the appearance, before        microlithiasis, of intrahepatic hyperechoic foci with or without        intrahepatic sludge.

In this syndrome, a deficiency in bile phospholipids is observed(abnormality of bile phospholipid secretion), limiting thesolubilization of cholesterol and leading to the precipitation thereofin the form of microcalculi in situ within the small bile ducts and, inextreme forms, within the entire biliary tree.

These microcalculi, which can be detected through examination of theliver by ultrasonography, exhibit the following characteristics:

-   -   they are observed in the form of multiple calculi which are        small in size (<2 mm), present in the intrahepatic bile ducts,    -   they are sensitive to treatment with ursodeoxycholic acid        (UDCA); this treatment makes it possible to obtain rapid        disappearance of the symptoms, unlike cholecystectomy, which        leads to a recurrence of the disease. This recurrence can,        however, be prevented by administering UDCA, and    -   they differ from gall bladder calculi by their small size, their        location and their sensitivity to treatment with UDCA.

The very large decrease in the concentration of bile phospholipids, andthe presence of cholesterol crystals sensitive to treatment with UDCA,combined with a mutation of the MDR3 gene, characterize this syndrome.This syndrome is now referred to as Low Phospholipid AssociatedCholelithiasis (LPAC).

The screening and the prevention of this pathological condition, whichis different from PFIC type 3, in individuals having personal or familyhistory of gall bladder lithiasis or of unexplained hepatic ailments(biological abnormalities relating to tests for cholestasis increase inserum activity of γ-glutamyltranspeptidases, transaminases) are crucial.Specifically, 10 to 20% of gravid cholestasis might, in fact, correspondto this syndrome, and its morbidity, including repeat abortions, mightthus be avoided in the case of good screening and effective prevention.

For this reason, the inventors gave themselves the aim of providing atest for screening this pathological condition, which differs fromprogressive familial intrahepatic cholestasis type 3 (PFIC3) bothclinically and biochemically, in such a way as to prevent its harmfuleffects, in particular in women of childbearing condition.

A subject-matter of the present invention is a method for the screeningof a hepatic syndrome, characterized in that it comprises at least thedetection, from a sample of nucleic acid extracted from peripheral bloodmononuclear cells, of at least one heterozygous mutation of the MDR3gene and/or of a homozygous mutation which does not destroy theexpression of the protein expressed by said gene withphosphatidylcholine transporter activity, in adult individualsassociating cholesterol microcholelithiasis and intrahepaticcholestasis.

For the purposes of the present invention, the expression “mutationwhich does not destroy the expression of the protein havingphosphatidylcholine transporter activity” is intended to mean bothmutations which lead to the expression of a protein having residualphosphatidyl transporter activity and mutations which decrease the levelof expression of the normal protein (mutation in the promoter forexample). These various mutations do not induce any hepatobiliarysymptoms in children, but the appearance, in young adults, of symptomsassociating cholesterol microcholelithiasis and intrahepaticcholestatis, induced by additional factors linked to the host and/or tothe environment.

In accordance with the invention, said MDR3 gene mutation is located inat least one of the following exons: 4, 5, 6, 8, 9, 10, 12, 14, 15, 16,17, 18, 19, 23 and/or 26.

More specifically:

-   -   in one aspect of the invention, said mutation is preferably        located in exon 6 and/or in exon 9 and/or in exon 12.    -   in another aspect of the invention, said mutation is preferably        located in exons 9, 10, 12, 14, 15, 17 or 18; indeed, as it        emerges from the obtained data, most mutations are localized in        the central part of the molecule, close to nucleotide binding        domain 1 (NBD1), or in adjacent transmembrane domains and        intracellular loops (see FIGS. 1 and 2). More precisely, 80% of        mutations are situated in regions encoded by exons 9 to 18,        which correspond approximately to 38% of the encoding region        (TM5 and 6; 3^(rd) intracellular loop including NBD1; TM7 and        8).    -   all the detected mutations (point mutations or other type of        mutations such as SNPs) are significantly related to LPAC, when,        based on a multivariate analysis (see example 3), able to define        a clinical score, said score being ≧2 (1 point if age at the        onset of symptoms was below 40 years, 1 point for recurrence        after cholecystectomy and 1 point for the presence of        intrahepatic spots).

According to an advantageous embodiment of said method, it comprises thedetection of a mutation selected from the group consisting of:

-   -   a mutation in exon 4 at nucleotide 175,    -   a mutation in exon 6 at nucleotide 495, 504 or 523,    -   a mutation in exon 8 at nucleotide 711,    -   a mutation in exon 9 at nucleotide 902 or 959,    -   a mutation in exon 10 at nucleotides 1007-1015 (insertion or        deletion)    -   a mutation in exon 12 at nucleotide 1327,    -   a mutation in exon 14 at nucleotide 1584,    -   a mutation in exon 15 at nucleotide 1772,    -   a mutation in exon 16 at nucleotide 1954,    -   a mutation in exon 17 at nucleotide 1973,    -   a mutation in exon 18 at nucleotides 2270-2273 (insertion)    -   a mutation in exon 19 at nucleotide 2363,    -   a mutation in exon 23 at nucleotide 2800 and    -   a mutation in exon 26 at nucleotide 3481.

More specifically, said method comprises the detection of a mutationselected from the group consisting of:

-   -   a heterozygous missense mutation in exon 6 at nucleotide 523:T/C        (TCG/CCG), leading to the amino acid mutation T175A; this amino        acid is located in a conserved amino acid sequence required for        the ATPase activity of the MDR3 protein,    -   a homozygous missense mutation in exon 9: at nucleotide 959:        C/T(TCC/TTC), leading to the amino acid mutation S320F, which is        located at the end of transmembrane domain 5 (TM5), and    -   a heterozygous mutation in exon 12: insertion of an adenine at        nucleotide 1327 (1327insA) in the first nucleotide-binding        domain (NBD1); this mutation causes a reading frame shift at        codon 443 and the appearance of a stop codon (nt 1339-1341),        leading to the expression of a 446 amino acid truncated protein.

These MDR3 gene mutations and others in patients with LPAC syndrome aresummed up in the following Tables I and II: TABLE I MDR3 gene pointmutations in patient with LPAC syndrome Location and Gene nucleotidePeptide Position change change Protein domain Status 6 495T→A Phe 165Ile 1^(st) intracellular loop Hetero 523A→G Thr 175 Ala between TMA-TM3Hetero 9 902T→C Met 301 Thr TM5 Hetero 959C→T Ser 320 Phe Homo 101007-1015insT 355 stop TM6 Hetero 1007-1015delT 341 stop TM6 Hetero 121327insA 447 stop Close to NBD1₁ Hetero 14 1584G→C Glu 528 Asp Close toNBD1₁ Hetero 15 1772T→A Leu 591 Gln 3^(rd) intracellular loop Homo 171973G→A Try 658 Stop 3^(rd) intracellular loop Hetero linker domain 182270-2273insT 793 Stop 4^(th) intracellular loop Hetero between TM8-TM919 2363G→T Arg 788 Glu 4^(th) intracellular loop Hetero between TM8-TM923 2800G→T Ala 934 Thr 5^(th) intracellular loop Homo between TM10-TM1126 3481C→T Pro 1161 Se Close to NBD2 HeteroNote:The A of ATG of the initiator Met codon was denoted as “nucleotide +1”

TABLE II Characterization of MDR3 gene SNPs and determination of themain allele frequency in patients with and without LPAC syndrome and incontrol subjects. SNP Localization Exon 4 Exon 5 Exon 6 Exon 8 Exon 16Nucleotide Change 175 C→T 342 T→C 504 T→C 711 A→T 1954 A→G Amino AcidLeu 59 Thr 114 Asn 168 Ile 237 Arg 652 Gly Most frequent allele CTG ACTAAC ATA AGG Mutation 21/24 24/24 16/30 21/24 23/24 LPAC (87.5%) (100%)(53.3%) (87.5%) (95.8%) Phenotype (Score ≧2) No mutation 27/28 27/283/28 27/28 27/28 LPCA (96.4%) (96.4%) (10.7%) (96.4%) (96.4%) phenotype(Score ≧2 No mutation 52/56 56/56 29/56 51/56 51/56 No LPAC (92.8%)(100%) (51.8%) (91.1%) (91.1%) phenotype (Score <2) Control 54/66 65/6636/66 50/66 61/66 subjects (81.8%) (98.5%) (54.5%) (89.3%) (92.4%)Bonferroni 0.05 0.99 0.001 0.14 0.99 Adjusted P-value (5 comparisons)

Said Table II indicates the most frequent allele to total number ofallele ratio for each tested SNPs in the different groups of patients.Nucleotide changes in SNPs are underlined.

Note: The A of ATG of the initiator Met codon was denoted as “nucleotide+1”

Thus five single-nucleotide polymorphisms (SNPs) in the coding region ofthe MDR3 gene may be identified in LPAC patients besides the pointmutations described here above in Table I. In all cases, a high score(as defined here above), i.e. ≧2 is obtained.

According to another advantageous embodiment of the method according tothe invention, it comprises:

-   -   a first step of amplification of a nucleic acid fragment, in        which at least one of said mutations is liable to be observed,        from the nucleic acid extracted from peripheral blood        mononuclear cells, using at least one primer selected from the        group consisting of the primers represented in the attached        sequence listing under the numbers SEQ ID NO: 1 to SEQ ID NO: 13        or in the hereafter Table III under the numbers SEQ NO: 15 to        SEQ ID NO:66, and

a second step of detection of the presence of at least one of saidmutations, using said amplification fragment(s) obtained. TABLE IIIIntronic primers for amplifying the different exons of MDR3 gene. Name(SEQ ID NO:) SEQUENCE MDR3-SENS2 GGAGAGGGTGTACTTGG (SEQ ID NO: 15)MDR3-AS2 AGGCATCAACCGATTTTTAC (SEQ ID NO: 16) MDR3-SENS3CTTTGTAGTACCTTCGACAG (SEQ ID NO: 17) MDR3-AS3 TTGTGCTCAAGCAACCCTCC (SEQID NO: 18) MDR3-SENS4 GAGGAGAAATTCCATTCCAC (SEQ ID NO: 19) MDR3-AS4CAACTCCCAAATTTTTACCC (SEQ ID NO: 20) MDR3-SENS5 TAAAAACCTGGCAATGCC (SEQID NO: 21) MDR3-AS5 AACTCTGTAATTGGAAATTATC (SEQ ID NO: 22) MDR3-SENS6CCATCATGGAGCTCATCACTT (SEQ ID NO: 23) MDR3-AS6 GCTGCCAGATGATCGATTTC (SEQID NO: 24) MDR3-SENS7 GTTTGTTGGATGTCTACTTC (SEQ ID NO: 25) MDR3-AS7CCTGAACAGGTACAAGTACG (SEQ ID NO: 26) MDR3-SENS8 GTGCCTTTAAACTTTTCTCC(SEQ ID NO: 27) MDR3-AS8 CGAGAAGGGTTAATATTAGG (SEQ ID NO: 28) MDR3-SENS9GCCGAGTGTGACTCGGAC (SEQ ID NO: 29) MDR3-AS9 GGTCTAACCACATGCTATTTTC (SEQID NO: 30) MDR3-SENS10 CTATGTTACATATACATCAC (SEQ ID NO: 31) MDR3-AS10GTACAACTTATTCAATGTAGTTG (SEQ ID NO: 32) MDR3-SENS11-12GACATTCCAGGTCCTATTTTTGG (SEQ ID NO: 33) MDR3-AS11-12GCTTGGTTCTTCCCACTTAC (SEQ ID NO: 34) MDR3-SENS13 GGTAGGATGTTTTTCATG (SEQID NO: 35) MDR3-AS13 CCTTTGAAGAATAAACTCAG (SEQ ID NO: 36) MDR3-SENS14GACAAAGCTCCATGTTGTC (SEQ ID NO: 37) MDR3-AS14 CTGTTTCTCAGCCCAGACTC (SEQID NO: 38) MDR3-SENS15 ATCCAAGTGCTTAACTGTG (SEQ ID NO: 39) MDR3-AS15GTATAGCATTCACTGGATC (SEQ ID NO: 40) MDR3-SENS16 TACATCCATTTGGAGACAC (SEQID NO: 41) MDR3-AS16 GCAAGGCTAAGAATTTC (SEQ ID NO: 42) MDR3-SENS17GCCTTTTCTATGTCTACAG (SEQ ID NO: 43) MDR3-AS17 AGAAGCAGCAGCTGATG (SEQ IDNO: 44) MDR3-SENS18 CTCAAGCCACTATTTATGAG (SEQ ID NO: 45) MDR3-AS18AGAATTTGGAAGCTCCATTAG (SEQ ID NO: 46) MDR3-SENS19 CAACTCATAACTTTGCTAC(SEQ ID NO: 47) MDR3-AS19 CATGCATATCGACATAACAATAAG (SEQ ID NO: 48)MDR3-SENS20 GGTCTCCCCTAAATTTCCTC (SEQ ID NO: 49) MDR3-AS20CAAGTGTGGGTATGCTACATG (SEQ ID NO: 50) MDR3-SENS21 GCTGGAGCGCATGCATTTG(SEQ ID NO: 51) MDR3-AS21 GTTGTAGTGGGCACAAA (SEQ ID NO: 52) MDR3-SENS22CTTGAACAGATTATGCCTTTGG (SEQ ID NO: 53) MDR3-AS22 TCCTAGTCACATCAAAAAGC(SEQ ID NO: 54) MDR3-SENS23 CTTAAACCCACTCGGCC (SEQ ID NO: 55) MDR3-AS23CACAGGAGTCATTTTTTTCCTAC (SEQ ID NO: 56) MDR3-SENS24 GACTTTCAAACATCATGGAG(SEQ ID NO: 57) MDR3-AS24 CTTATCCTGTAGCTATAATC (SEQ ID NO: 58)MDR3-SENS25 CTGGCACCAGAACTATACC (SEQ ID NO: 59) MDR3-AS25ATTATGACAATATTGGTTGGGC (SEQ ID NO: 60) MDR3-SENS26 GAAGCTGCTGACACCC (SEQID NO: 61) MDR3-AS26 GAAGTGCCTTGTCCAAGTTG (SEQ ID NO: 62) MDR3-SENS27AATAGAACTGTCAACTGTTAAGC (SEQ ID NO: 63) MDR3-AS27 TTTTCCCCCTGTGCTTG (SEQID NO: 64) MDR3-SENS28 GATTAGAAAGGTAACATTTTC (SEQ ID NO: 65) MDR3-AS28GGGTCTTCTAAATTGATC (SEQ ID NO: 66)

According to the invention, said nucleic acid fragment on which theamplification is performed is selected from the group which consists inmRNA, cDNA and genomic DNA.

According to an advantageous arrangement of this embodiment of themethod, the first amplification step consists of direct PCRamplification of at least one of exons 6, 9 and 12 of the genomic DNAwith the following primers, located in the flanking intronic regions:

-   -   primers SEQ ID NO: 1 and SEQ ID NO: 2, which make it possible to        amplify a 321 pb fragment covering exon 6,    -   primers SEQ ID NO:3 and SEQ ID NO:4, which make it possible to        amplify a 210 pb fragment covering exon 9, and    -   primers SEQ ID NO:5 and SEQ ID NO:6, which make it possible to        amplify a 154 pb fragment covering exon 12.

According to another advantageous arrangement of this embodiment of themethod, the first amplification step consists of RT-PCR amplification ofthe mRNAs by two overlapping PCRs, using two pairs of primers, selectedfrom the group consisting of the following pairs:

-   -   first pair: SEQ ID NO: 7 and SEQ ID NO: 8    -   second pair:    -   SEQ ID NO: 9 and SEQ ID NO: 10    -   SEQ ID NO: 11 and SEQ ID NO: 10    -   SEQ ID NO: 12 and SEQ ID NO: 10, and    -   SEQ ID NO: 13 and SEQ ID NO: 10.

More specifically, for the detection from the mRNA, the method accordingto the invention advantageously comprises:

a first PCR amplification of 2100 pb fragment (fragment 1) correspondingto exons 1 to 16 of the human MDR3 gene, with the pair of primers:5′-CCTGCCAGACACGCGCGAGGTTC-3′ (SEQ ID NO: 7) and5′-CTTCAAGTCCATCGGTTTCCACATC-3′, (SEQ ID NO: 8) and

-   -   a second amplification of fragment 1, using various pairs of        primers, capable respectively of amplifying a fragment        comprising exon 6, a fragment comprising exon 9 and a fragment        comprising exon 12.

Even more specifically:

a 1746 pb fragment (fragment 2) comprising exon 6 is amplified with thefollowing pair of primers: 5′-CCTTGTCGCTGCTAAATCC-3′ (SEQ ID NO: 9) and5′-GGCTCTTCTGACACATTTGTG-3′; (SEQ ID NO: 10)

a 1483 pb fragment (fragment 3) comprising exon 9 is amplified with thefollowing pair of primers: (SEQ ID NO: 11) and SEQ ID NO: 105′-GGAATTGGTGACAAGGTTGG-3′;

a 1162 pb fragment (fragment 4) comprising exon 9 is amplified with thefollowing pair of primers: (SEQ ID NO: 12) and SEQ ID NO: 105′-GCTATTTCAGCAAACATTTCCATGG-3′;

an 823 pb fragment (fragment 5) comprising exon 12 is amplified with thefollowing pair of primers: (SEQ ID NO: 13) and SEQ ID NO: 105′-GCTAACGTCAAGATCTTGAAGG-3′.

According to another advantageous arrangement of this embodiment of themethod, the first amplification step consists of direct PCRamplification of at least one of exons 6, 9, 10, 12, 14, 15, 17, 18, 19,23, and 26 and further of at least one of exons 4, 5, 8 and/or 16 of thegenomic DNA with the primers, located in the flanking intronic regions,as specified in the hereabove Table III (SEQ ID NO: 15 to 66).

According to yet another advantageous arrangement of this embodiment ofthe method, the second step of detection of the presence of at least oneof said mutations using the amplified fragment is carried out, asappropriate, using one of the following methods:

-   -   sequencing    -   enzyme restriction or    -   techniques of the PCR/PCR/LDR reaction.

When the mutation results in the appearance or disappearance of arestriction site in the amplified fragment, the mutations are detectedby digestion with the enzyme recognizing said restriction site, comparedwith a control which does not exhibit said mutation.

For example:

-   -   digestion of the 210 pb amplification fragment as defined above        with the HinfI enzyme: detection of a 210 pb fragment        corresponds to the presence of wild-type allele, whereas        detection of two fragments (142 pb and 48 pb) corresponds to the        presence of the mutated allele;    -   digestion of the 210 pb amplification fragment as defined above        with the BamHI enzyme: detection of a 210 pb fragment        corresponds to the presence of the mutated allele and detection        of two fragments corresponds to the presence of the wild-type        allele.

When the mutation cannot be detected by enzyme digestion, the fragmentamplified by PCR is sequenced according to conventional techniques; byway of example, mention may be made of the dideoxynucleotide technique.

When the mutation to be detected corresponds to the insertion or to thedeletion of a small sequence, said mutation can be detected usingtechniques of the PCR/PCR/LDR type, which are based on detecting theligation of allele-specific primers using a thermostable ligase (Faviset al., Nature Biotech., 2000, 18, 561-564).

Such a method may advantageously be used to evaluate the risk ofappearance of a syndrome associating intrahepatic cholestasis andcholesterol micro-cholelithiasis in families at risk of cholesterollithiasis and/or having unexplained hepatic biological abnormalities.

When a mutation, as defined above, is detected in individuals at risk,said evaluation method can advantageously also comprise searching forintrahepatic microcrystals of cholesterol, measuring the cholesterolsaturation index of the bile, determining the cholesterol/bilephospholipids ratio, assaying bile phospholipids, and testing againstursodeoxycholic acid.

The diagnosis may be considered to be positive for the abovementionedsyndrome when one of the abovementioned mutations is screened,associated with the presence of microcholelithiasis, a very low level ofbile phospholipids and a cholesterol saturation index of the bile ofgreater than 1.

Another subject-matter of the present invention is also the use of thevarious primers as defined above, for the screening of a syndromeassociating (i) intrahepatic hyperechoic foci with or withoutintrahepatic sludge or microlithiasis and further on cholesterolmicrocholelithiasis, intrahepatic cholestasis and (ii) at least onemutation of the MDR3 gene.

Another subject-matter of the present invention is also a kit for thescreening of a syndrome associating (i) intrahepatic hyperechoic fociwith or without intrahepatic sludge or microlithiasis and further oncholesterol microcholelithiasis, intrahepatic cholestasis and (ii) atleast one mutation of the MDR3 gene, characterized in that it comprisesat least two primers as defined above, capable of detecting a mutationof the human MDR3 gene, as defined above.

Another subject-matter of the present invention is also a kit forevaluating the risk of appearance of a syndrome associating (i)intrahepatic hyperechoic foci with or without intrahepatic sludge ormicrolithiasis and further on cholesterol microcholelithiasis,intrahepatic cholestasis and (ii) at least one mutation of the MDR3gene, in a population at risk, characterized in that it comprises,besides the primers capable of detecting the mutation of the MDR3 geneas defined above, reagents for assaying cholesterol and bilephospholipids.

Preferably, the reagents are enzymatic reagents.

Another subject-matter of the present invention is also the use ofursodeoxycholic acid for preparing a medicinal product intended to treata hepatic syndrome associating (i) intrahepatic hyperechoic foci with orwithout intrahepatic sludge or microlithiasis and further on acholesterol microcholelithiasis, intrahepatic cholestasis and (ii) atleast one mutation of the MDR3 gene.

BRIEF DESCRIPTION OF DRAWINGS

Besides the above arrangements, the invention also comprises otherarrangements which will emerge from the following text, which refers toexamples of implementation of the present invention and also to theattached drawings in which:

FIG. 1 illustrates the location of the various mutations on the cDNAencoding the MDR3 protein. Exons (Ex) 6 to 12 are represented by whiteboxes, the black boxes correspond to transmembrane domains (TM) 2 to 6and the hashed box represents the nucleotide-binding domain (NBD1). Thelocation of the various mutations is represented by arrows. ICL1corresponds to the intracellular loop of the MDR3 protein, which isessential for the ATPase activity of this protein.

FIG. 2 illustrates the localization of the mutations in the differentdomains of the MDR3 protein. The gray boxes correspond to thetransmembrane domains of the protein. TMD1 and 2 correspond to the twosymmetrical regions containing the TM domains 1-6 and 7-12,respectively. NBD 1 and 2 correspond to the nucleotide binding domainsand the black stars correspond to the mutations tha have been identifiedin patients presenting LPAC. The figures in the stars indicate thenumber of patients having the corresponding mutation.

FIG. 3 illustrates as the study profile of example 3.

FIG. 4 illustrates MDR3 gene mutations in LPAC and other human liverdiseases: genotype-phenotype relationship.

EXAMPLE 1 Materials and Methods

1—Characteristics of the Patients Included in the Study

Six patients with no family history of PFIC were studied: five women(patients 1, 2, 4, 5 and 6) and one man (patient 3). They exhibit:chronic intrahepatic cholestasis, cholesterol cholelithiasis,intrahepatic microcholelithiasis as attested by echography and anabnormality of the MDR3 gene; treatment with ursodeoxycholic acid (UDCA)leads to complete disappearance of the symptoms and standardizing of theenzymatic constants of the liver. In the five women, this syndromeappeared during pregnancy or after treatment with oral contraceptives.

Analysis of the bile shows: an oversaturation of cholesterol, a lowphospholipid level and the presence of cholesterol crystals.

2—Preparation of DNA and RNA

Circulating blood mononuclear cells are isolated by centrifugation(Histopaque-1119, Sigma Diagnostics). The DNA is extracted from 5×10⁶lymphocytes using the QIAamp kit (Qiagen-GmbH, Germany). The total RNAis extracted from 5×10⁶ peripheral blood nuclear cells (kit fromEurobio, les Ulis, France) in accordance with the manufacturer'sinstructions. The RNA and DNA concentrations are quantified byspectrophotometry.

3—Amplification of the MDR Gene Transcripts by RT-PCR

A reverse transcription is carried out in a reaction volume of 20 μlcontaining 500 mM of dNTP, 10 mM of DTT, 0.5 U.ml⁻¹ of RNasine (Madison,Wis.), 5 mM of random hexamers and 10 μg.ml⁻¹ of reverse transcriptase(Gibco BRL, Bethesda, Md.). The RNA extracts are heated for 5 minutes at70° C. and cooled on ice before being added (1 μg) to the reactionmixture and incubated for one hour at 37° C.

The cDNA obtained (5 μl, 0.25 μg) is amplified by the PCR method usingsynthetic oligonucleotide primers (Genosys, Cambridge, UK). A first PCR,producing the 2100 pb fragment 1, is carried out in a reaction medium of25 μl containing 10 mM of Tris-HCl, pH 8.3, 1.5 mM of magnesiumchloride, 0.001% of gelatin, 0.05 U of Taq DNA polymerase, 350 μM ofeach dNTP and 0.5 μM of each primer (sense and antisense) as defined inTable IV below: TABLE IV Sequence of the primers and size of thefragments obtained by RT- PCR amplification of the MDR3 gene Frag-Position Product ment Pairs of PCR primers (5′→3′) Position (exon) size(pb) 1 CCTGCCAGACACGCGCGAGGTTC (SEQ ID NO: 7)   32-2078  (1-16) 2100CTTCAAGTCCATCGGTTTCCACATC (SEQ ID NO: 8) 2 CCTTGTCGCTGCTAAATCC (SEQ IDNO: 9)  296-2041  (5-16) 1746 GGCTCTTCTGACACATTTGTG (SEQ ID NO: 10) 3GGAATTGGTGACAAGGTTGG (SEQ ID NO: 11)  559-2041  (9-16) 1483GGCTCTTCTGACACATTTGTG (SEQ ID NO: 10) 4 GCTATTTCAGCAAACATTTCCATGG (SEQID NO: 12)  880-2041  (9-16) 1162 GGCTCTTCTGACACATTTGTG (SEQ ID NO: 10)5 GCTAACGTCAAGATCTTGAAGG (SEQ ID NO: 13) 1219-2041 (11-16)  823GGCTCTTCTGACACATTTGTG (SEQ ID NO: 10) 6 GAGGCCAACGCCTATGAG (SEQ ID NO:14) 1522-2041 (13-16)  520 GGCTCTTCTGACACATTTGTG (SEQ ID NO: 10)

The pair of primers for the amplification of fragment 1 has already beendescribed (J M. De Vree et al., mentioned above).

The primary PCR amplification is carried out under the followingconditions: an initial denaturation step at 94° C. for five minutes isfollowed by 35 cycles alternating a denaturation step at 94° C. forthirty seconds, a primer hybridization step at 68° C. for thirtyseconds, and an extension step at 72° C. for two minutes.

Overlapping PCRs producing five overlapping fragments (fragments 2, 3,4, 5 and 6) are carried out by adding 2 μl of the amplification productof the primary PCR to a secondary PCR reaction mixture (50 μl). Thereaction medium is identical to that described above (for the primers,see Table IV).

The secondary PCR amplification is carried out under the followingconditions: an initial denaturation step at 94° C. for five minutes isfollowed by 35 cycles alternating a denaturation step at 94° C. forthirty seconds, a primer hybridization step at 60° C. for thirtyseconds, and the extension step at 72° C. for 90 seconds.

Negative controls are carried out on RNA samples amplified in theabsence of reverse transcriptase. The amplification products areseparated by electrophoresis in a 1.5% agarose gel containing ethidiumbromide. The amplified DNA fragments are visualized under an ultravioletlamp.

4—PCR Amplification of Exons 6, 7, 9 and 12 of the MDR3 Gene

Exons 6, 9 and 12 are amplified by PCR using primers located in theflanking intronic regions and using genomic DNA as matrix:

-   -   a 321 pb fragment covering exon 6 is amplified using the        following primers:    -   sense primer 5′-CTACTCAGGATTGGGTGCTGG-3′ (SEQ ID NO: 1) and    -   antisense primer 5′-GCTAGAACATGGCTGCCAG-3′ (SEQ ID NO: 2);    -   a 210 pb fragment covering exon 9 is amplified using the        following primers:    -   sense primer 5-CCCTCTCATTTTTCTGGTAG-3′ (SEQ ID NO: 3) and    -   antisense primer 5′-GTTAGGAGAACTACTTACTG-3′ (SEQ ID NO: 4);    -   a 154 pb fragment covering exon 12 is amplified using the        following primers:    -   sense primer 5′-CCTTACAGATCTTGAAGGGC-3′ (SEQ ID NO: 5) and    -   antisense primer 5′-GCTTGGTTCTTCCCACTTAC-3′ (SEQ ID NO: 6).

The PCR reaction is carried out in a final volume of 50 μl, in thepresence of 100 ng of genomic DNA and of 1.5 mM of MgCl₂. The primerhybridization step is carried out at 60° C. (exon 7), 50° C. (exon 9)and 56° C. (exon 12), and the other steps are identical to those set outabove for the cDNA, with the exception of the number of cycles (30instead of 35). The amplification products are separated byelectrophoresis in a 2% agarose gel containing ethidium bromide. Theamplified DNA fragments are visualized under an ultraviolet lamp.

5—Analysis of Mutations

The amplification products are sequenced (dideoxyterminator kit) inaccordance with the manufacturer's (Perkin-Elmer) instructions. Thesequencing reactions are carried out in a Perkin-Elmer thermocycler 9600using the same primers as for the amplification of the exons (see 4-).The extension products are separated from the nonincorporatednucleotides and from the primers by column centrifugation (quick spinTM, Boehringer Mannheim), and they are then dried and resuspended in 4μl of deionized formamide containing 50 mM of EDTA, pH8. These productsare then heated for 2 minutes at 90° C., transferred onto ice andimmediately loaded onto a denaturing 6% polyacrylamide gel. Themigration is carried out at 30 W for 12 h (automated DNA sequencer,model ABI 373 A).

6—Restriction Analysis

The exon-9 amplification fragments are digested (15 μl) for 6 hours at37° C. with Hinf I or BamH I (2 IU, Biolabs), and the fragments obtainedare visualized on 2% agarose gel.

7—Bile Composition

A polarized light microscope is used to detect the presence ofcholesterol crystals in samples of fresh bile.

The concentration of total bile salts is measured by an enzymatictechnique using 3α-hydroxysteroid dehydrogenase (Enzabile, Nycomed,Oslo, Norway). The content of total phospholipids is determined by anenzymatic method in the presence of phospholipase D and choline oxidase.The cholesterol is determined by an enzymatic reaction with cholesteroloxidase or cholesterol esterase. The cholesterol saturation indices forthe hepatic bile samples are calculated from Carey's critical tables (J.Lipid Res., 1978, 19, 945-955).

EXAMPLE 2 Results

The six patients mentioned above were studied.

1—Morphology and Histopathology

Ultrasonography (US) makes it possible to detect multiple microcalculiin the intrahepatic bile ducts in the six patients.

A liver biopsy sample obtained in patients 3 and 4 shows moderate portalinflammation and proliferation of the bile ducts without any damage tosaid ducts. Extensive fibrosis is also observed in patient 4.

As regards patient 3, many biliary microcalculi visible on the X-raywere extracted from the intrahepatic and extrahepatic bile ducts.Biochemical analysis of these calculi show that they consist essentiallyof cholesterol.

A cholangiography was performed in patient 5, and shows localizedsclerozing cholangitis characterized by irregular left intrahepatic bileducts exhibiting alternating regions of stenosis and regions of sacculardilation.

2—Composition of the Hepatic Bile

The bile lipid composition of the hepatic bile was studied using samplesobtained by radioscopy of the duodenum (patients 2 and 3) or using a Ttube (patients 5 and 6). The results obtained in the untreated patientsor the patients receiving treatment with UDCA are given respectively inTables V and VI below. TABLE V Bile lipid composition of the hepaticbile from untreated patients Normal Parameters measured values* Case 2Case 5 Case 6 Bile lipid classes (mol %) Bile salts (BS) 72.6 72.1 57.394.2 Phospholipids (PL) 20.6 18.7 25.3 1.4 Cholesterol (CH) 7.8 9.1 17.44.5 Cholesterol 1.1 1.98 20.9 ND saturation index (CSI) Lipid ratiosCH/PL 0.38 0.49 0.70 3.21 PL/BS 0.29 0.26 0.44 0.01 CH/BS 0.10 0.13 0.300.05 Concentration of 15.6 6.39 1.77 0.30 bile PL*The normal values are derived from Lamont et al., (Progress in LiverDiseases, Boyer et al. Ed., W. B. Saunders Company, Philadelphia, 1992,165-191).

TABLE VI Bile lipid composition of the hepatic bile from patientsreceiving treatment with UDCA Normal Parameters measured values* Case 5Case 6 Bile lipid classes (mol %) Bile salts (BS) 72.6 47.5 72Phospholipids (PL) 20.6 35.7 19.7 Cholesterol (CH) 7.8 16.8 8.2Cholesterol 1.1 4.0 1.8 saturation index (CSI) Lipid ratios CH/PL 0.380.47 0.42 PL/BS 0.29 0.75 0.27 CH/BS 0.10 0.35 0.11 Concentration ofbile PL 15.6 7.77 6.03*The normal values are derived from Lamont et al. (mentioned above).

Analysis of the bile samples by microscopy shows the presence of manycholesterol monohydrate crystals.

-   -   All the freshly taken bile samples derived from patients not        treated with UDCA have the following three characteristics:        -   1°) a high cholesterol saturation index (CSI),        -   2°) a high cholesterol/phospholipid (CH/PL) ratio, and        -   3°) a low or a very low concentration of phospholipids (PL),            in comparison with the normal concentrations described (Lee            et al., 1986, Gastroenterology, 90, 677-686; Lamont et al.,            mentioned above).

In patient 5, the large increase in the cholesterol/bile salts (CH/BS)ratio indicates that hypersecretion of cholesterol is responsible forthe increase in the cholesterol saturation of the bile.

In patient 6, the large decrease in the phospholipids/bile salts (PL/BS)and cholesterol/bile salts (CH/BS) ratios indicates that, in this case,it is a decrease in the secretion of bile salts which is responsible forthe increase in the cholesterol saturation of the bile.

-   -   After treatment with UDCA, a large decrease in the cholesterol        saturation index is observed (patients 5 and 6), associated with        an increase in the phospholipid concentration of the bile.        Consequently, a decrease in cholesterol/bile salts ratio is        observed in patient 5, whereas the low values for the        cholesterol/bile salts and phospholipids/bile salts ratios        become normal again in patient 6.

3—Analysis of the Mutations of the MDR3 Gene

The sequence of fragments 2, 3, 4, 5 and 6 obtained by RT-PCR wasanalyzed, and the mutations observed are as follows:

Insertion

In patients 2 and 3, the insertion of a single nucleotide in position1327 of exon 12 is detected, in the form of a heterozygous mutation.This mutation leads to a modification of the open reading frame at codon443, which leads to the appearance of a premature stop codon and theproduction of a 446 amino acid truncated protein.

Missense Mutation

A missense mutation at codon 320 of exon 9 (Ser₃₂₀ (TCC)→Phe₃₂₀(TTC),nucleotide 959 T→C) is present in the homozygous state in independentaffected individuals derived from non-blood-related families (patients 1and 4). However, the unaffected individuals of these two families areheterozygous for this substitution. This mutation is not detected oneither of the alleles of the 63 independent controls (126 chromosomes),which demonstrates that this mutation does not correspond to apolymorphism of the MDR3 gene.

A missense mutation at codon 175 of exon 6 (Thr₁₇₅(ACG)→Ala₁₇₅ (GCG),nucleotide 523 A→G) is present in the heterozygous state in patient 5.This mutation is not detected on either of the alleles of 51 independentcontrols (102 chromosomes), which demonstrates that this mutation doesnot correspond to a polymorphism of the MDR3 gene.

The sequencing of the fragments amplified from the genomic DNA confirmsthe homozygocity of patients 1 and 4 for the mutation S320F, theheterozygocity of patients 2 and 3 for the mutation 1327insA and theheterogocity of patient 5 for the mutation T175V.

Digestion of the amplified fragments with restriction enzymes confirmsthe presence of the mutation S320F in patient 1 (homozygous transitionT→C) and in the members of the same family (heterozygous transitionT→C), and also the absence of this mutation in normal nonrelatedindividuals.

No mutation was detected in exons 6, 9 and 12 of patient 6, who probablyhas a mutation in the promoter.

EXAMPLE 3 Second Study

Patients

32 patients, who were referred specifically for MDR3 gene analysisbecause of clinical history compatible with the syndrome that isdescribed hereabove (e.g. having a symptomatic cholelithiasis with atleast one of the following criteria: age at onset of symptoms less than40, symptomatic cholelithiasis, recurrence after cholecystectomy,intraheptic hyperechoic foci with a topography compatible with lipiddeposits along the luminal surface of the intrahepatic biliary tree withor without intrahepatic sludge or microlithiasis, familial history ofcholelithiasis in 1^(st) degree relatives, clinical history ofintrahepatic cholestasis of pregnancy), were studied.

A second group of twenty-eight other patients presenting with a classicform of gallstone disease revealed by a single episode (or a secondepisode in one case) of typical biliary pain or cholecystitis thatjustified cholecystectomy constituted the control group. None of thesepatients had inflammatory biliary diseases (such as primary biliarycirrhosis, primary sclerosing cholangitis), fibrocystic liver disease(Caroli's disease), congenital hepatic fibrosis, Rendu-Osler, cysticfibrosis, protoporphyria, total parenteral nutrition, obesity (BMI≧30),biliary infection or infestation with parasites (including HIVinfection), malabsorption (including ileal resection) and somatostatinsynthetic analogs or hypocholesterolemic agent therapy, who wereexcluded by clinical examination and appropriate investigationsincluding the results of anti-mitochondrial and anti-perinuclearantineutrophil cytoplasmic autoantibodies, ultrasonography,echoendoscopy, magnetic resonance cholangiography, T-tubecholangiography when a surgical treatment was performed, ERCP and, infew cases, liver biopsy. None of these patients had a medical history ofprogressive familial intrahepatic cholestasis during childhood andpatients suffering from black pigment stones, hemolysis or cirrhosiswere also excluded.

A third group comprises 33 consecutive patients without history ofcholelithiasis and presenting with chronic cholestasis (primary biliarycirrhosis n=4, primary sclerosing cholangitis n=4), diverse acute orchronic liver diseases (chronic HBV or HCV hepatitis, non-alcoholicsteatohepatitis (NASH), drug-induced hepatitis) (n=15) or a classicgenetic hemochromatosis associated with C282Y homozygous mutation in theHFE gene (n=10) constituted a second control group. Based on previousdata, the clinical feature characteristic of the syndrome were collectedfor the 60 patients with cholelithiasis as summed up in the followingTable VII. TABLE VII Patient characteristics Patients (n) 60 Age (years± SD) 38.1 ± 14.4 Gender (M/F) 15/45 (n) (%) Age at onset of symptoms<40 years 37/60 62 Symptomatic cholelithiasis 60/60 100 Recurrence aftercholecystectomy 28/60 47 Cholecystectomy 54/60 90 Intrahepatichyperechoic foci * 32/60 53 Medical history of ICP 15/46 33 Increasedserum GGT activity ** 22/60 37 Familial history of cholelithiasis 21/5042 Prophylactic UDCA treatment 25/39 64UDCA denotes ursodeoxycholic acid.ICP denotes intrahepatic cholestasis of pregnancy.* e.g. consisting intrahepatic hyperechoic foci with a topographycompatible with lipid deposits along the luminal surface of theintrahepatic biliary tree with or without intrahepatic sludge ormicrolithiasis.** at the time of genotype determination

To determine the prevalence of the MDR3 mutations and single-nucleotidepolymorphisms (SNPs), and the clinical factors predictive of thesemutations in patients with symptomatic cholelithiasis, the entire MDR3sequence in all 93 patients (see FIG. 3) was analysed. Written, informedconsent was obtained from all participants.

Mutation Screening

Genomic DNA was obtained from peripheral white blood cells usingstandard procedures. We designed specific primers to amplify exons andsplice junctions, using the Massachusetts Institute of Technology website. The sequences of these intronic primers are those described inTable III.

Each PCR reaction contained 200 ng genomic DNA, with each primer at aconcentration of 0.4 μM, 0.08 μM deoxynucleoside triphosphates(Pharmacia, Piscataway, N.J.), 1.5 mM magnesium chloride and 1.25 U Taqpolymerase. PCR products were purified on sephadex column and sequencedusing Big Dye Terminator Chemistry (Applied Biosystems). Identificationand localization of MDR3 gene mutations and SNPs was assessed bysequence comparisons using Phred Phrap Consed Software.

Statistical Analysis

Fisher's exact test was used to compare proportions. All tests were atthe 5% level, and reported P-values were two-sided. A multivariable,logistic regression model was used to select a set of clinical featurespredictive of a point mutation at the MDR3 locus. The multivariablemodel was selected by minimising the Akaike information criterion, astandard statistical procedure for selecting variables to be included aspredictors (Burnham K. et al., Model selection and multi-modelinterference. NY, Springler Verlag, 2002). When comparing SNPs at theMDR3 locus, the first species risk was adjusted to the number ofcomparisons (=number of SNPs) according to the Bonferroni rule. Allstatistical analyses were performed using the R software (version 1.5.1)(Ihaka R. et al., J. Computational and Graphical Statistics, 1996, 5,299-314).

Results

1. Clinical Phenotype Associated with MDR3 Gene Mutations

Table VIII: Unadjusted Odds-Ratio for the Presence of a Mutation at theMDR3 Locus in Cholelithiasis Patients

Clinical Criteria OR CI 95% P-value Familial history of 5.4 [1.2, 29.4]0.01 cholelithiasis in 1^(st) degree relatives Increased serum GGT 1.1[0.3, 4.1] 1 activity at the time of genotype determination History ofICP 4.9 [1.1, 24.0] 0.02 Intrahepatic hyperchoic foci 12.4 [2.4, 126.0]0.0005 Recurrence after cholecystectomy 18.9 [3.6, 193.7] <0.0001 Age atonset of symptoms <40 7.8 [1.5, 77.8] 0.008 Gender (Male vs Female) 0.8[0.1, 3.4] 1

Among the 32 patients suspected of having the syndrome, 18 (56%)presented a point mutation in the MDR3 gene while none of the 28patients with a classical form of cholelithiasis and none of the 33patients without cholelithiasis had mutation in this gene (p<0.001 andp<0.0001, respectively). For the whole population of 60 patients withsymptomatic cholelithiasis, the unadjusted odds-ratios for the presenceof a MDR3 gene mutation in this population are presented in Table VIII.Multivariate analysis showed that three independent factors werepredictive of a mutation at the MDR3 locus: a recurrence of symptomsafter cholecystectomy (adjusted OR=8.5), intrahepatic hyperechoic foci(adjusted OR=6.1), and age <40 years (adjusted OR=3.0).

Based on the multivariate analysis, a clinical score indicative of thepresence of a MDR3 mutation was defined as follows: 1 point if age atthe onset of symptoms was below 40 years, 1 point for recurrence aftercholecystectomy and 1 point for the presence of intrahepatic spots.

All patients with a point mutation scored higher than 2 (mean ±SE:2.7±0.5). By contrast, patients without point mutations had scoredranging from 0 to 3 (mean ±SE: 1.2±1.1) and could be divided into twogroups (see FIG. 3): one group of patients scoring ≧2 (n=14) and anothergroup scoring <2 (n=28). The score was therefore highly sensitive forthe presence of a mutation (Se=100%, CI 95% [85%-100%]) but was notspecific in this sample of patients (Sp=67%, CI 95% [52%-81%]).

2. Identification of MDR3 Gene Mutation in Patients with Cholelithiasis(Table 3)

Patients were screened for mutations in the MDR3 gene using PCRamplification and DNA sequencing of exons 2 to 28 and all splicejunctions. 14 heterozygous and homozygous point mutation were identifiedamongst these 18 patients, all of them with a predictive score ≧2. Noneof these mutations was detected in a control panel of one hundred andforty chromosomes, thus demonstrating that they did not correspond topolymorphisms.

The mutations included as in example 2, three 1 bp-insertions and one 1bp-deletion, resulting in a frameshift predicted to cause prematuremessenger RNA termination, a loss of protein function and tensingle-nucleotide substitutions including one null mutation. Affectedpatients with heterozygous mutations exhibited 1 bp-insertion, 1bp-deletion, nonsense mutations or missense mutations, while affectedpatients with homozygous mutations demonstrated only missense mutations.Most mutations were localized as mentioned here above in the centralpart of the molecule, close to nucleotide binding domain 1 (NBD1), or inadjacent transmembrane domains and intracellular loops (see FIG. 3).Eighty percent of mutations were indeed situated in regions encoded byexons 9 to 18, which corresponded approximately to 38% of the encodingregion (TM 5 and 6; 3^(rd) intracellular loop including NBD1, TM 7 and8).

In contrast, no point mutations could be detected in 14 patients with ahigh score (≧2) and 28 patients with a low score (<2) (see FIG. 3).Furthermore, MDR3 gene sequence analysis in the 33 patients withoutcholelithiasis demonstrated two heterozygous missense mutationsaffecting non-conserved amino acids (Arg590Gln and Gly742Ser).

3. Identification of MDR3 Gene SNPs in Patients with Cholelithiasis (SeeTable II)

In order to search for a MDR3 defect even in patients with a high scorebut no point mutation, we investigated the frequency of MDR3 gene SNPsin these patients (see FIG. 3). The previously described Arg652Glypolymorphism was detected with a similar frequency (about 5%) inpatients with and without MDR3 mutations and in control subjects(Jacquemin E. et al., Gastroenterology, 2001, 120, 1448-1458).

Five novel single-nucleotide polymorphisms (SNPs) in the coding regionof the MDR3 gene have also been identified (see Table II here above).The allele frequency of the most frequent variant (e.g. 504 T→C, AAC)was only 10.7% in the group of LPAC syndrome patients with a high scorebut no MDR3 point mutations, while it reached 54.5% in control subjects,51.8% in patients with cholelithiasis and a low score and 53.3% inpatients with LPAC syndrome and MDR3 gene mutations (Bonferroni adjustedP-value <0.001). Other SNPs were more rare and were detected at similarfrequencies in the different patient groups and in control subjects.

The mutation screening described in the invention method was unable todetect major DNA rearrangements, and nor did the analysis include thepromoter or other potential regulation regions of the gene. Despitethese limitations, the unexpected low frequency of the 504 C>T variant(Leu 168 Leu) in the subgroup of patients with LPAC syndrome but withoutan MDR3 gene point mutation suggests that some of them may have beenhemizygous for this region, so that a large deletion in exon 6 could beimplicated in the disease. Such synonymous single-nucleotidepolymorphisms located in coding regions, although seeminglytranslationally silent, could also have a profound influence onalternative splicing and potentially lead to exon skipping or aberrantsplicing. Alternatively, defects in other regions of the gene or inother genes may also be involved, and some evidence from animal studieshas pointed to Abcb 11 (previously called the bile salt export pump,BSEP) or Abcc 2 (previously referred to as multidrug resistance relatedprotein 2, or MRP2) as other possible candidate genes underlyingsusceptibility to cholelithiasis or phospholipid secretion disruption.

In summary, the results strongly support the role of an MDR3 gene defectin LPAC syndrome and replace it in the context of MDR3 gene-associatedliver diseases (see FIG. 4).

All references, patents and patents applications cited herein areexpressly incorporated by reference into the present specification intheir entireties.

1. A method of screening for a hepatic syndrome associated with one ormore of intrahepatic hyperechoic foci with or without intrahepaticsludge or microlithiasis, cholesterol microcholelithiasis andintrahepatic cholestasis, wherein said method comprises detecting in asample of nucleic acid extracted from peripheral blood mononuclear cellsof a subject, at least one heterozygous mutation of the MDR3 gene or atleast one homozygous mutation of the MDR3 gene, or both, wherein saidmutation or mutations do not destroy the expression of the proteinexpressed by said gene with phosphatidylcholine transporter activity,wherein the presence of said mutation or mutations in the nucleic acidof said subject is an indicator of susceptibility to said hepaticsyndrome associated with one or more of intrahepatic hyperechoic fociwith or without intrahepatic sludge or microlithiasis, cholesterolmicrocholelithiasis and intrahepatic cholestasis.
 2. The method of claim1, wherein at least one of said mutations is located within any one orall of the exons selected from the group consisting of exon 6, exon 9and exon
 12. 3. The method of claim 2, wherein at least one of saidmutations is selected from the group consisting of a mutation in exon 9at nucleotide 959 and a mutation in exon 12 at nucleotide
 1327. 4. Themethod of claim 3, wherein the mutation located at nucleotide 959 is ahomozygous missense mutation C/T (TCC/TTC), leading to the amino acidmutation S320F, and the mutation located at nucleotide 1327 correspondsto the insertion of an adenine (1327insA).
 5. The method of claim 1,wherein said MDR3 gene mutation or mutations are located in at least oneof the following exons: 4, 5, 6, 8, 9, 10, 12, 14, 15, 16, 17, 18, 19,23 or
 26. 6. The method of claim 5, wherein said mutation or mutationsare located in at least one of exons 9, 10, 12, 14, 15, 17 or
 18. 7. Themethod of claim 5, wherein said method comprises the detection of amutation or mutations selected from the group consisting of: a) amutation in exon 4 at nucleotide 175, b) a mutation in exon 6 atnucleotide 495, 504, c) a mutation in exon 8 at nucleotide 711, d) amutation in exon 9 at nucleotide 902 or 959, e) a mutation in exon 10 atnucleotides 1007-1015 (insertion or deletion), f) a mutation in exon 12at nucleotide 1327, g) a mutation in exon 14 at nucleotide 1584, h) amutation in exon 15 at nucleotide 1772, i) a mutation in exon 16 atnucleotide 1954, j) a mutation in exon 17 at nucleotide 1973, k) amutation in exon 18 at nucleotides 2270-2273 (insertion), l) a mutationin exon 19 at nucleotide 2363, m) a mutation in exon 23 at nucleotide2800 and n) a mutation in exon 26 at nucleotide
 3481. 8. The method ofclaim 5, wherein said method comprises: a) amplifying nucleic acidextracted from peripheral blood mononuclear cells, using at least oneprimer selected from the group consisting of SEQ ID NO: 1 through SEQ IDNO: 13 and SEQ ID NO: 15 through SEQ ID NO:66, and b) detecting thepresence of at least one of said mutations in the amplified product ofstep (a).
 9. The method of claim 8, wherein the first amplification stepconsists of direct PCR amplification of at least one of exons 6, 9 and12 of the genomic DNA with the following primers, located in theflanking intronic regions: a) primers SEQ ID NO: 1 and SEQ ID NO: 2,which make it possible to amplify a 321 bp fragment covering exon 6, b)primers SEQ ID NO: 3 and SEQ ID NO: 4, which make it possible to amplifya 210 bp fragment covering exon 9, and c) primers SEQ ID NO: 5 and SEQID NO: 6, which make it possible to amplify a 154 bp fragment coveringexon
 12. 10. The method of claim 8, wherein the first amplification stepconsists of RT-PCR amplification of mRNAs by two overlapping PCRs, usingtwo pairs of primers selected from the group consisting of the followingpairs: a) SEQ ID NO: 7 and SEQ ID NO: 8 b) SEQ ID NO: 9 and SEQ ID NO:10 c) SEQ ID NO: 11 and SEQ ID NO: 10 d) SEQ ID NO: 12 and SEQ ID NO:10, and e) SEQ ID NO: 13 and SEQ ID NO:
 10. 11. The method of claim 8,wherein the first amplification step consists of direct PCRamplification of at least one of exons 6, 9, 10, 12, 14, 15, 17, 18, 19,23, and 26 and further of at least one of exons 4, 5, 8 and 16 of thegenomic DNA with the primers, located in the flanking intronic regions,wherein said primers are selected from the group consisting of any oneor more of SEQ ID NO: 15 through SEQ ID NO:
 66. 12. The method of claim8, wherein the second step of detection of the presence of at least oneof said mutations using the amplified fragment is carried out using oneof the following methods: sequencing, enzyme restriction or techniquesof the PCR/PCR/LDR type.
 13. The method of claim 9, wherein thedetection step is carried out by digestion of the 210 bp amplificationfragment with the HinfI enzyme.
 14. The method of claim 9, wherein thedetection step is carried out by digestion of the 210 bp amplificationfragment with the BamHI enzyme.
 15. The method of claim 1, wherein saidsubject is determined to be at risk of developing cholesterol lithiasisor having unexplained hepatic biological abnormalities.
 16. A method ofscreening for a syndrome associated with one or more of intrahepatichyperechoic foci with or without intrahepatic sludge or microlithiasis,cholesterol microcholelithiasis and intrahepatic cholestasis, whereinsaid method comprises contacting a nucleic acid sample extracted fromperipheral blood mononuclear cells of a subject with primers selectedfrom the group consisting of SEQ ID NO: 1 through SEQ ID NO: 13 and SEQID NO: 15 through SEQ ID NO: 66, wherein said contacting results in theidentification of at least one mutation of the MDR3 gene and whereinsaid identification is an indicator of susceptibility to developing saidsyndrome.
 17. A kit for the screening of a hepatic syndrome associatedwith at least one mutation of the MDR3 gene, wherein said kit comprisesat least two primers selected from the group consisting of SEQ ID NO: 1through SEQ ID NO: 13 and SEQ ID NO: 15 to SEQ ID NO:
 66. 18. A kit forevaluating a population at risk of developing a hepatic syndromeassociated with at least one mutation of the MDR3 gene comprising, inaddition to two primers selected from the group consisting of SEQ ID NO:1 to SEQ ID NO: 13 or SEQ ID NO: 15 to SEQ ID NO: 66, reagents forassaying cholesterol and bile phospholipids.
 19. A method of treating apatient having a syndrome associated with at least one of intrahepatichyperechoic foci with or without intrahepatic sludge or microlithiasis,cholesterol microcholelithiasis and intrahepatic cholestasis and atleast one mutation of the MDR3 gene, wherein said method comprisesadministering to said patient a pharmaceutical composition comprisingursodeoxycholic acid.
 20. The method of claim 1, wherein said subject isa young adult.